TCR Pipeline Results Summary

Summary

Library	Total Reads in FASTQ	Pct Reads Passing Quality Filter	Pct CellLabel UMI Aligned Uniquely
N22 TCR	83,671,440	38.03%	39.55%

Putative Cells

85,988

Bioproduct Type	Aligned Reads By Type	Mean Reads per Cell	Mean Molecules per Cell
mRNA	3,875,539	32.62	29.79
Sample Tags	18,520	0.05	-
VDJ TCR	542,230	2.93	0.18

Graphs

No graphs available. (Usually due to too few or too many cells)

Sequence Quality

Library	Total Reads in FASTQ	Pct Read Pair Overlap	Pct Reads Too Short	Pct Reads Low Base Quality	Pct Reads High SNF	Pct Reads Filtered Out	Total Reads After Quality Filtering
N22 TCR	83,671,440	31.56%	58.09%	0.21%	3.67%	61.97%	31,817,242

Library Quality

Library	Total Filtered Reads	Pct Q30 Bases in Filtered R2	Pct CellLabel UMI	Pct CellLabel UMI Aligned Uniquely	Pct Reads Useful
N22 TCR	31,817,242	62.94%	48.18%	39.55%	13.94%

Alignment Categories

Library	CellLabel UMI Reads	Annotated Transcriptome Pct	Introns Pct	Intergenic Regions Pct	Antisense Pct	Not Unique Pct	Ambiguous Pct	No Feature Pct	AbSeq Pct	Sample Tag Pct	VDJ TCR Pct	VDJ BCR Pct	Unaligned Pct
N22 TCR	15,329,454	1.53%	23.75%	17.47%	13.22%	21.5%	0.92%	0.04%	0%	0.12%	3.54%	0%	17.92%

Reads and Molecules

Bioproduct Type	Aligned Reads By Type	Total Raw Molecules	Total RSEC Molecules	Mean Raw Sequencing Depth	Mean RSEC Sequencing Depth	Sequencing Saturation
mRNA	3,875,539	3,560,922	3,556,521	1.09	1.09	14.56

Cells

Putative Cells

85,988

Basic Algorithm Cells

85,988

Bioproduct Type	Pct Reads from Putative Cells	Mean Reads per Cell	Median Reads per Cell	Mean Molecules per Cell	Median Molecules per Cell	Mean Bioproducts per Cell	Median Bioproducts per Cell	Total Bioproducts Detected
mRNA	72.37%	32.62	27	29.79	25	18.45	16	24,220

Sample Multiplexing

Valid Reads aligned to Sample Tags

18,520

Pct Reads from Putative Cells

72.6%

Sample Tag	Sample Names	Raw Reads	Pct of Raw Reads	Cells Called	Pct of Putative Cells Called	Raw Reads in Called Cells	Mean Reads per Called Cell
All_Tags	-	18,520	100%	85,988	100%	3,894	0.05
SampleTag01_flex	-	1,658	8.95%	1	0%	220	220
SampleTag02_flex	-	3,814	20.59%	72	0.08%	890	12.36
SampleTag03_flex	-	1,550	8.37%	0	0%	0	0
SampleTag04_flex	-	1,652	8.92%	25	0.03%	284	11.36
SampleTag05_flex	-	2,789	15.06%	49	0.06%	588	12
SampleTag06_flex	-	3,841	20.74%	64	0.07%	1,306	20.41
SampleTag07_flex	-	1,363	7.36%	16	0.02%	168	10.5
SampleTag08_flex	-	1,848	9.98%	30	0.03%	438	14.6
SampleTag09_flex	-	Not_Detected	-	-	-	-	-
SampleTag10_flex	-	Not_Detected	-	-	-	-	-
SampleTag11_flex	-	Not_Detected	-	-	-	-	-
SampleTag12_flex	-	2	0.01%	0	0%	0	0
SampleTag13_flex	-	Not_Detected	-	-	-	-	-
SampleTag14_flex	-	Not_Detected	-	-	-	-	-
SampleTag15_flex	-	1	0.01%	0	0%	0	0
SampleTag16_flex	-	Not_Detected	-	-	-	-	-
SampleTag17_flex	-	Not_Detected	-	-	-	-	-
SampleTag18_flex	-	1	0.01%	0	0%	0	0
SampleTag19_flex	-	1	0.01%	0	0%	0	0
SampleTag20_flex	-	Not_Detected	-	-	-	-	-
SampleTag21_flex	-	Not_Detected	-	-	-	-	-
SampleTag22_flex	-	Not_Detected	-	-	-	-	-
SampleTag23_flex	-	Not_Detected	-	-	-	-	-
SampleTag24_flex	-	Not_Detected	-	-	-	-	-
Multiplet	-	-	-	0	0%	-	-
Undetermined	-	-	-	85,731	99.7%	-	-

VDJ

Reads

Chain Category	Reads Cellular Aligned to VDJ	Reads Contig Assembled	Reads VDJ Annotated	Reads Putative	Reads Corrected	Pct Reads Corrected	Mean Reads Corrected per Putative Cell
TCR	542,230	524,574	316,056	294,636	252,297	48.1%	2.93
TCR Alpha	-	41,853	19,590	18,409	14,838	35.45%	0.17
TCR Beta	-	461,099	289,144	269,286	231,120	50.12%	2.69
TCR Delta	-	2,309	781	752	721	31.23%	0.01
TCR Gamma	-	19,313	6,541	6,189	5,618	29.09%	0.07

Molecules and Dominant Contigs

Chain Category	Mean Molecules Corrected per Putative Cell	Molecules VDJ Annotated	Molecules Corrected	Dominant Contigs Mean Nucleotide Length	Dominant Contigs Pct Full Length	Dominant Contigs Pct With CDR3
TCR	0.18	23,949	15,754	300	28.46%	99.59%
TCR Alpha	0.03	3,991	2,866	347	42.46%	99.28%
TCR Beta	0.14	19,059	12,167	290	25.55%	99.77%
TCR Delta	0	259	231	309	26.03%	94.52%
TCR Gamma	0.01	640	490	299	27.34%	99.28%

Cell Type VDJ Metrics:

Cell Type	Number cells	TCR Paired Chains Any Pct	TCR Paired Chains Full Pct
B	74	16.22%	8.11%
Dendritic	14	0%	0%
Monocyte classical	84,080	0.31%	0.12%
Monocyte nonclassical	20	0%	0%
Natural killer	403	10.92%	4.71%
T CD4 memory	411	30.9%	19.22%
T CD4 naive	761	12.22%	6.18%
T CD8 memory	154	25.97%	12.99%
T CD8 naive	51	9.8%	3.92%
T gamma delta	20	35%	5%

Cell Type	TCR Alpha Pct Cells Positive	TCR Beta Pct Cells Positive	TCR Delta Pct Cells Positive	TCR Gamma Pct Cells Positive
B	16.22%	22.97%	1.35%	1.35%
Dendritic	0%	0%	0%	0%
Monocyte classical	0.6%	3.99%	0.04%	0.09%
Monocyte nonclassical	0%	5%	0%	0%
Natural killer	10.17%	18.61%	2.98%	4.71%
T CD4 memory	30.9%	44.04%	1.7%	3.89%
T CD4 naive	12.61%	22.08%	0.39%	1.31%
T CD8 memory	25.97%	38.96%	5.19%	7.79%
T CD8 naive	9.8%	23.53%	0%	0%
T gamma delta	30%	35%	25%	25%

Cell Type	TCR Alpha Pct Cells Full Length	TCR Beta Pct Cells Full Length	TCR Delta Pct Cells Full Length	TCR Gamma Pct Cells Full Length
B	8.11%	21.62%	0%	0%
Dendritic	0%	0%	0%	0%
Monocyte classical	0.19%	0.72%	0.01%	0.02%
Monocyte nonclassical	0%	0%	0%	0%
Natural killer	5.21%	12.16%	1.24%	1.99%
T CD4 memory	21.17%	34.55%	0.49%	1.46%
T CD4 naive	7.23%	15.11%	0.13%	0.26%
T CD8 memory	13.64%	31.17%	1.95%	4.55%
T CD8 naive	3.92%	13.73%	0%	0%
T gamma delta	15%	25%	5%	10%

Cell Type	TCR Alpha Mean Molecules per Cell	TCR Beta Mean Molecules per Cell	TCR Delta Mean Molecules per Cell	TCR Gamma Mean Molecules per Cell
B	0.59	2.76	0.01	0.01
Dendritic	0	0	0	0
Monocyte classical	0.01	0.08	0	0
Monocyte nonclassical	0	0.05	0	0
Natural killer	0.33	1.73	0.18	0.23
T CD4 memory	2.06	5.68	0.08	0.23
T CD4 naive	0.58	2.11	0.02	0.06
T CD8 memory	0.94	4.53	0.14	0.32
T CD8 naive	0.16	2.35	0	0
T gamma delta	0.9	2.9	1.4	1.95

Pipeline Inputs

Pipeline version: 2.0

Pipeline Start Time: Wed Jan 03 2024 14:06:20 GMT+0000 (Greenwich Mean Time)

AbSeq Reference:

No value

Reference Files Archive:

RhapRef_Human_WTA_2023-02.tar.gz

Supplemental Reference:

No value

Targeted Reference:

No value

Reads:

N22_TCR_S1_R1_001.fastq.gz
N22_TCR_S1_R2_001.fastq.gz

Multiplexing Settings

Sample Tags Version: flex
Tag Names:

VDJ Settings

VDJ Species Version: humanTCR

Putative Cell Calling Settings

Enable Refined Putative Cell Calling: False
Exact Cell Count: No value
Expected Cell Count: No value
Putative Cell Call: mRNA

Exclude Intronic reads: False

Generate Bam output: True

Metric Alerts

(Under development)

For the [N22_TCR] library, the [Pct_Read_Pair_Overlap] metric value of 31.56% is greater than 20% (Sequencing Quality section, WTA VDJ assay threshold).

May be due to short products in the library prep.
Look for evidence of this in QC traces and contact customer support for suggestions on additional cleanups.

For the [N22_TCR] library, the [Pct_Reads_Too_Short] metric value of 58.09% is greater than 10% (Sequencing Quality section, WTA VDJ assay threshold).

May be due to short products in the library prep.
Look for evidence of this in QC traces and contact customer support for suggestions on additional cleanups.

For the [N22_TCR] library, the [Pct_CellLabel_UMI] metric value of 48.18% is less than 70% (Library Quality section, WTA VDJ assay threshold).

May be due to poor sequencing or library quality, or incorrect reference panel.
Check Q30 scores and QC traces, and confirm that the appropriate transcriptome reference and complete AbSeq reference was used.
This can also occur if V1 and V2 beads are combined in a single library.

For the [N22_TCR] library, the [Pct_CellLabel_UMI_Aligned_Uniquely] metric value of 39.55% is less than 40% (Library Quality section, WTA VDJ assay threshold).

May be due to poor sequencing or library quality; check QC traces and Illumina metrics to confirm sequencing was high quality.
Ensure that correct reference panel was used.
Ensure reads from long-read libraries have been trimmed before running the pipeline.

For the [N22_TCR] library, the [Unaligned_Pct] metric value of 17.92% is greater than 10% (Alignment Categories section, WTA assay threshold).

May occur in experiments with low cell input. This can be addressed with additional cleanups for low cell input libraries (included in the protocol).
Can also result from low cell viability or poor sequencing or library quality.
This is expected if samples such as nuclei containing a high proportion of unprocessed transcripts are being run.

Pipeline Version: 2.0

Wed Jan 03 2024 14:06:20 GMT+0000 (Greenwich Mean Time)

Summary

Graphs

Sequence Quality

Library Quality

Alignment Categories

Reads and Molecules

Cells

Sample Multiplexing

VDJ

Pipeline Inputs

Metric Alerts

(Under development)